Peptide-based compounds

ABSTRACT

This invention relates to new peptide-based compounds and their use in therapeutically effective treatments as well as for diagnostic imaging techniques. More specifically the invention relates to the use of such peptide-based compounds used as targeting vectors that bind to receptors associated with angiogenesis, in particular the αvβ3 integrin receptor. Such contrast agents may thus be used for diagnosis of for example malignant diseases, heart diseases, inflammation-related diseases, rheumatoid arthritis and Kaposi&#39;s sarcoma. Moreover such compounds may also be used in therapeutic treatment of these diseases.

This application is a continuation application of internationalapplication number PCT/NO01/00390 filed Sep. 25, 2001, which claimsbenefit of priority to application number 20004795 filed Sep. 26, 2000.PCT/NO01/00390 is also a Continuation-in-Part to U.S. provisional patentapplication No. 60/259,919 filed Jan. 5, 2001, the entire disclosures ofwhich are hereby incorporated by reference.

This invention relates to new peptide-based compounds and their use intherapeutically effective treatments as well as for diagnostic imagingtechniques. More specifically the invention relates to the use of suchpeptide-based compounds used as targeting vectors that bind to receptorsassociated with angiogenesis, in particular the αvβ3 integrin receptor.Such contrast agents may thus be used for diagnosis of for examplemalignant diseases, heart diseases, inflammation-related diseases,rheumatoid arthritis and Kaposi's sarcoma. Moreover such compounds mayalso be used in therapeutic treatment of these diseases.

BACKGROUND OF THE INVENTION

New blood vessels can be formed by two different mechanisms:vasculogenesis or angiogenesis. Angiogenesis is the formation of newblood vessels by branching from existing vessels. The primary stimulusfor this process may be inadequate supply of nutrients and oxygen(hypoxia) to cells in a tissue. The cells may respond by secretingangiogenic factors, of which there are many; one example, which isfrequently referred to, is vascular endothelial growth factor (VEGF).These factors initiate the secretion of proteolytic enzymes which breakdown the proteins of the basement membrane, as well as inhibitors whichlimit the action of these potentially harmful enzymes. The otherprominent effect of angiogenic factors is to cause endothelial cells tomigrate and divide. Endothelial cells which are attached to the basementmembrane, which forms a continuous sheet around blood vessels on thecontralumenal side, do not undergo mitosis. The combined effect of lossof attachment and signals from the receptors for angiogenic factors isto cause the endothelial cells to move, multiply, and rearrangethemselves, and finally to synthesise a basement membrane around the newvessels.

Angiogenesis is prominent in the growth and remodeling of tissues,including wound healing and inflammatory processes. Tumors must initiateangiogenesis when they reach millimeter size in order to keep up theirrate of growth. Angiogenesis is accompanied by characteristic changes inendothelial cells and their environment. The surface of these cells isremodeled in preparation for migration, and cryptic structures areexposed where the basement membrane is degraded, in addition to thevariety of proteins which are involved in effecting and controllingproteolysis. In the case of tumors, the resulting network of bloodvessels is usually disorganised, with the formation of sharp kinks andalso arteriovenous shunts. Inhibition of angiogenesis is also consideredto be a promising strategy for antitumor therapy. The transformationsaccompanying angiogenesis are also very promising for diagnosis, anobvious example being malignant disease, but the concept also showsgreat promise in inflammation and a variety of inflammation-relateddiseases, including atherosclerosis, the macrophages of earlyatherosclerotic lesions being potential sources of angiogenic factors.These factors are also involved in re-vascularisation of infracted partsof the myocardium, which occurs if a stenosis is released within a shorttime.

Further examples of undesired conditions that are associated withneovascularization or angiogenesis, the development or proliferation ofnew blood vessels are listed in Table 1 below. Reference is also made inthis regard to WO98/47541.

Diseases and indications associated with angiogenesis are e.g. differentforms of cancer and metastasis, e.g. breast, skin, colorectal,pancreatic, prostate, lung or ovarian cancer.

Other diseases and indications are inflammation (e.g. chronic),atherosclerosis, rheumatoid arthritis and gingivitis.

Further diseases and indications associated with angiogenesis arearteriovenous alformations, astrocytomas, choriocarcinomas,glioblastomas, gliomas, hemangiomas (childhood, capillary), hepatomas,hyperplastic endometrium, ischemic myocardium, Kaposi sarcoma, maculardegeneration, melanoma, neuroblastomas, occluding peripheral arterydisease, osteoarthritis, psoriasis, retinopathy (diabetic,proliferative), scleroderma, seminomas, solid tumor formation andulcerative colitis.

Angiogenesis involves receptors which are unique to endothelial cells.The integrin αvβ3 is one of the receptors that is known to be associatedwith angiogenesis. Stimulated endothelial cells appear to rely on thisreceptor for survival during a critical period of the angiogeneicprocess, as antagonists of the ∀v∃3 integrin receptor/ligand interactioninduce apoptosis and inhibit blood vessel growth.

The integrin αvβ3 is a member of a family of transmembrane proteins thatact as receptors through which cells can adhere to the extracellularmatrix. Integrins are heterodimeric molecules in which the α- andβ-subunits penetrate the cell-membrane lipid bilayer. The α-subunit hasfour Ca²⁺ binding domains on its extracellular chain, and the β-subunithas a number of extracellular cysteine-rich domains.

Many ligands (eg. fibronectin) involved in cell adhesion contain thetripeptide sequence arginine-glycine-aspartic acid (RGD). The RGDsequence appears to act as a primary recognition site between theligands presenting this sequence and receptors on the surface of cells.It is generally believed that secondary interactions between the ligandand receptor enhance the specificity of the interaction. These secondaryinteractions might take place between moieties of the ligand andreceptor that are immediately adjacent to the RGD sequence or at sitesthat are distant from the RGD sequence.

RGD peptides are known to bind to a range of integrin receptors and havethe potential to regulate a number of cellular events of significantapplication in the clinical setting. (Ruoslahti, J. Clin. Invest., 87:1-5 (1991)). Perhaps the most widely studied effect of RGD peptides andmimetics thereof relate to their use as anti-thrombotic agents wherethey target the platelet integrin GpIIbIIIa.

Inhibition of angiogenesis in tissues by administration of either anαvβ3 or αvβ5 antagonist has been described in for example WO 97/06791and WO95/25543 using either antibodies or RGD containing peptides. EP578083 describes a series of mono-cyclic RGD containing peptides and WO90/14103 describes RGD-antibodies. Haubner et al. in the J. Nucl. Med.(1999); 40: 1061-1071 describe a new class of tracer for tumourtargeting based on monocyclic RGD containing peptides. Biodistributionstudies using whole-body autoradiographic imaging revealed however thatthe ¹²⁵I-labelled peptides had very fast blood clearance rates andpredominantly hepatobiliary excretion routes resulting in highbackground noise.

Cyclic RGD peptides where the RGD moiety is constrained by bridgingacross the ends of the tripeptide sequence have also been described inWO98/54347 and WO95/14714. Peptides derived from in vivo biopanning(WO97/10507) have been used for a variety of targeting applications. Thesequence CDCRGDCFC (RGD-4C), with unidentified bridge positions, hasbeen used to target drugs such as doxirubicin (WO98/10795), nucleicacids and adenoviruses to cells (see WO99/40214, WO99/39734, WO98/54347,WO98/54346, U.S. Pat. No. 5,846,782).

The efficient targeting and imaging of integrin receptors associatedwith angiogenesis in vivo demands a selective, high affinity RGD basedvector which is chemically robust and stable. Furthermore, the route ofexcretion is an important factor when designing imaging agents in orderto reduce problems with background. These stringent conditions are metby the structures containing discrete bridges described in the presentinvention.

SUMMARY OF THE INVENTION

In view of the needs of the prior art, the present invention providesnew peptide-based compounds and their use in therapeutically effectivetreatments as well as for diagnostic imaging techniques. Moreover, theinvention relates to the use of such peptide-based compounds used astargeting vectors that bind to receptors associated with angiogenesis,in particular the αvβ3 integrin receptor. Such contrast agents may thusbe used for diagnosis of for example malignant diseases, heart diseases,inflammation-related diseases, rheumatoid arthritis and Kaposi'ssarcoma. Additionally, such compounds may also be used in therapeutictreatment of these diseases.

A compound of general formula (I):

whereinr=0 or 1p=0 or 1and r+p=1when r=0 then R₁ is —(CH₂)_(n)—CO— or —(CH₂)_(n)—C₆H₄—CO— where n=1, 2,3, 4 or 5,when r=1 then R₁ is or one or more bridge-forming amino acids.X₁=a bond or 1, 2, 3, 4 or 5 amino acids, or an amino acid derivatizedwith a carbohydrate moiety, or an amino acid functionalised with aspacer or linker and/or a chelate binding or capable of binding areporter suitable for in vivo imaging,X₂ and X₄ are independently cysteine, homocysteine or other amino acidscapable of forming a cyclising bond such as aspartic acid and lysine,X₃ is arginine, N-methylarginine or an arginine mimetic,X₅ is a hydrophobic amino acid,X₆ is an amino acid capable of forming a cyclising bond,X₇ is a bond or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or a spaceror linker, optionally allowing for labelling with multiple chelates asdefined by X₈, and optionally comprising one or more ethylene glycolunits or any other spacer component, andX₈ is a chelate binding to, or capable of binding a metal radionuclideor any other reporter suitable for in vivo imaging, or is —NH₂ or isabsent,q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, andone of the bridges (between R₁ and X₂ or between X₄ and X₆) comprises adisulphide bond.

Further, the present invention provides a pharmaceutical compositioncomprising an effective amount of a compound of general formula (I) oran acid addition salt thereof to enhance image contrast for in vivoimaging or for therapeutic treatment, together with one or morepharmaceutically acceptable adjuvants, excipients or diluents.

Still further, the present invention provides a method of generatingenhanced images of a human or non-human animal body which comprisesadministering to the body a pharmaceutical composition of the presentinvention and generating an image of at least a part of the body.

Even still further, the present invention provides a method ofmonitoring the effect of treatment of a human or non-human animalsubject with a drug to combat a condition associated with angiogenesiswhich comprises administering to the subject a compound of formula I anddetecting the uptake of the compound, the administration and detectionoptionally but preferable being effected repeatedly, e.g. before, duringand after treatment with the drug.

The present invention yet further provides the use of a compound forformula I in the manufacture of therapeutic compositions and in methodsof therapeutic or prophylactic treatment of the human or non-humananimal body and/or for the manufacture of a contrast medium for use in amethod of diagnosis involving administration of the contrast medium toan animate subject and generation of an image or at least part of thesubject.

The present invention even further yet provides a process for thepreparation of a compound of formula I, which includes the steps ofconjugating a vector V to a compound detectable in a diagnostic imagingprocedure or a chelant compound and if necessary metalliting chelantgroups in the resultant conjugate with a metal ion detectable in adiagnostic imaging procedure.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

Viewed from one aspect the invention provides new peptide-basedcompounds as defined by formula I. These compounds have utility asvectors with affinity for the integrin αvβ3 and comprising a linear RGDsequence flanked by two discrete bridges where one or both of thebridges is a disulphide bridge. Such vectors have now shown unexpectedactivity by improved binding/efficacy compared to known linear RGDpeptides. These new peptide-based compounds may be used intherapeutically effective treatments as well as for imaging purposes.

Formula I thus defines peptide-based compounds used as vectors (V)having affinity for the integrin αvβ3. However, depending on thedefinitions of R₁ and X₁₋₈, formula I also includes compounds of theformula “V-L-R” where

V is the vector, L is a linker moiety or a bond, and R is a detectablemoiety (reporter), e.g. detectable in an imaging procedure, such as invivo imaging of the human or vascularized non-human animal body (e.g.mammalian, avian or reptilian body), wherein said compound ischaracterised by the general formula (I)

wherein

r=0 or 1

p=0 or 1

and r+p=1

when r=0 then R₁ is —(CH₂)₂—CO— or —(CH₂)_(n)—C₆H₄—CO— where n=1, 2, 3,4 or 5,

when r=1 then R₁ is or one or more bridge-forming amino acids,preferable cysteine and preferably the bridge between R₁ and X₂ containsa thioether or a disulphide bond, and when r=1 (and thus p=0) then R₁ isparticularly preferably cysteine and forms a disulphide bridge with X₂,

X_(i)=a bond or 1, 2, 3, 4 or 5 amino acids, or an amino acidderivatized with a carbohydrate moiety, or an amino acid functionalisedwith a spacer or linker and/or a chelate binding or capable of binding areporter suitable for in vivo imaging, preferably a metal radionuclide,preferably X₁ is aspartic acid, tyrosine, tyrosine-aspartic acid orlysine,

X₂ and X₄ are independently cysteine, homocysteine or other amino acidscapable of forming a cyclising bond such as aspartic acid and lysine,

X₃ is arginine, N-methylarginine or an arginine mimetic,

X₅ is a hydrophobic amino acid, preferably phenylalanine, tyrosine,iodotyrosine (most preferably 3-iodo-tyrosine), diiodotyrosine ornaphthylalanine,

X₆ is an amino acid capable of forming a cyclising bond, preferablecysteine or homocysteine,

X₇ is a bond or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, preferablyglycine, or a spacer or linker, optionally allowing for labelling withmultiple chelates as defined by X₈, and optionally comprising one ormore ethylene glycol units or any other spacer component, and

X₈ is a chelate binding to, or capable of binding a metal radionuclideor any other reporter suitable for in vivo imaging, or is —NH₂ or isabsent,

q is 0, 1, 2, 3, 4, 5, 6, 7 or 8, and

one of the bridges (between R₁ and X₂ or between X₄ and X₆) comprises adisulphide bond.

The vector components of the vector-chelate conjugates described hereinhave in some aspects of the invention no free amino- or carboxy-termini.Such termini introduce into these compounds a significant increase inresistance against enzymatic degradation and as a result they have anincreased in vivo stability as compared to many known free peptides.

The invention relates preferably to a compound of formula (I), furtherdefined by formula (II)

wherein

X₁′ is 1, 2 or 3 amino-acids, most preferably aspartic acid, tyrosine,tyrosine-aspartic acid, lysine or acetyl-lysine,

X₃ is as defined in formula I;

X₅′ is phenylalanine, tyrosine, 3-iodo-tyrosine or naphthylalanine;

X₇′ is a bond, glycine, or a O-bis(aminoethyl)ethylene glycol spacer,preferably X, is glycine; and

X₈′ is a chelate binding to a metal radionuclide wherein the structureof the chelate is

or any other N₃S or bis-oxime type of these chelates, and

one bridge comprises a thio bond and the other bridge comprises adisulphide bond as shown in formula II.

In particularly preferred embodiments of the compounds of generalformula (II), X₃ is N-methyl-arginine and/or X₅′ is naphthylalanine.

The reporter, R, may be attached to V (via L) at any suitable pointwithin X₁ and/or X₇. Preferably, the point of attachment is chosen suchthat the biological activity of V or the binding affinity of V for itstarget is not substantially or not significantly reduced (in comparisonwith the biological activity of V or the binding affinity of V withoutR). Most preferably, R is attached to V via X₁ and/or X₇.

As used herein the term ‘amino acid’ refers in its broadest sense toproteogenic L-amino acids, D-amino acids, chemically modified aminoacids, N-methyl, C-methyl and amino acid side-chain mimetics andunnatural amino acids such as naphthylalanine.

The term ‘cyclising bond’ refers to any combination of amino acids (orwith amino acid and —(CH₂)_(n)—CO— or —(CH₂)_(n)—C₆H₄—CO—) withfunctional groups which allows for the introduction of a bridge. Somepreferred examples are disulphides, disulphide mimetics such as the—(CH₂)_(n)— carba bridge, thioacetal, thioether bridges (cystathione orlanthionine) and bridges containing esters and ethers.

Some preferred embodiments of the compounds of formula (I) areillustrated by Compounds 1-5 below:

In most cases, it is preferred that the amino-acids in the vector V arein the L-form. However, in some embodiments of the invention one, two,three or more of the amino-acids in the vector V are preferably in theD-form. The inclusion of such D-form amino-acids can have a significanteffect on the serum stability of the vector. Reference is particularlymade in this regard to vectors having D-tyrosine at position X₁.

The invention also provides a pharmaceutical composition comprising aneffective amount (e.g. an amount effective to enhance image contrast inin vivo imaging and/or for therapeutic treatment) of a compound ofgeneral formula (I) or an acid addition salt thereof, together with oneor more pharmaceutically acceptable adjuvants, excipients or diluents.

As mentioned above, the compounds of formula I may comprise vector,linker and reporter moieties. A linker moiety may serve to link onevector to one reporter; alternatively it may link together more than onevector and/or more than one reporter. Likewise a reporter or a vectormay be linked to more than one linker. Use in this way of a plurality ofreporters (e.g. several linker-reporter moieties attached to one vectoror several reporters attached to one linker itself attached to onevector) may enable the detectability of the contrast agent to beincreased (e.g. by increasing its radioopacity, echogenicity orrelaxivity) or may enable it to be detected in more than one imagingmodality. Use in this way of a plurality of vectors may increase thetargeting efficiency of the contrast agent or may make the contrastagent able to target more than one site, e.g. different receptors for anagent which has receptor heterogeneity.

Linker

A wide variety of linkers can be used, including biodegradable linkersand biopolymers.

The linker component of the contrast agent is at its simplest a bondbetween the vector and reporter moieties. More generally however thelinker will provide a mono- or multi-molecular skeleton covalently ornon-covalently linking one or more vectors to one or more reporters,e.g. a linear, cyclic, branched or reticulate molecular skeleton, or amolecular aggregate, with in-built or pendant groups which bindcovalently or non-covalently, e.g. coordinatively, with the vector andreporter moieties or which encapsulate, entrap or anchor such moieties.Thus linking of a reporter unit to a desired vector may be achieved bycovalent or non-covalent means, usually involving interaction with oneor more functional groups located on the reporter and/or vector.Examples of chemically reactive functional groups which may be employedfor this purpose include amino, hydroxyl, sulfhydryl, carboxyl, andcarbonyl groups, as well as carbohydrate groups, vicinal diols,thioethers, 2-aminoalcohols, 2-aminothiols, guanidinyl, imidazolyl andphenolic groups.

It will be appreciated that functional groups in the reporter and/orvector may if desired be converted to other functional groups prior toreaction, e.g. to confer additional reactivity or selectivity.

Vector-reporter coupling may also be effected using enzymes aszero-length crosslinking agents; thus, for example, transglutaminase,peroxidase and xanthine oxidase have been used to produce crosslinkedproducts. Reverse proteolysis may also be used for crosslinking throughamide bond formation.

Non-covalent vector-reporter coupling may, for example, be effected byelectrostatic charge interactions, through chelation in the form ofstable metal complexes or through high affinity binding interaction.

A vector which is coupled to a peptide, lipooligosaccharide orlipopeptide linker which contains a element capable of mediatingmembrane insertion may also be useful.

Coupling may also be effected using avidin or streptavidin, which havefour high affinity binding sites for biotin. Avidin may therefore beused to conjugate vector to reporter if both vector and reporter arebiotinylated.

So-called zero-length linking agents, which induce direct covalentjoining of two reactive chemical groups without introducing additionallinking material may, if desired, be used in accordance with theinvention.

Most commonly, however, the linking agent will comprise two or morereactive moieties, e.g. as described above, connected by a spacerelement. The presence of such a spacer permits bifunctional linkers toreact with specific functional groups within a molecule or between twodifferent molecules, resulting in a bond between these two componentsand introducing extrinsic linker-derived material into thereporter-vector conjugate.

The nature of extrinsic material introduced by the linking agent mayhave a critical bearing on the targeting ability, pharmacokinetics andgeneral stability of the ultimate product. Thus it may be desirable tointroduce labile linkages, e.g. containing spacer arms which arebiodegradable or chemically sensitive or which incorporate enzymaticcleavage sites. Alternatively the spacer may include polymericcomponents, e.g. to act as surfactants and enhance the stability of theagent. The spacer may also contain reactive moieties, e.g. as describedabove to enhance surface crosslinking.

Spacer elements may also comprise macromolecular structures such asdextran and poly(ethyleneglycols), usually referred to as PEGs. Inaddition to spacer elements, PEGs may also be used to modify the in vivocharacteristics of the vectors.

The major mechanism for uptake of particles by the cells of thereticuloendothelial system (RES) is opsonisation by plasma proteins inblood; these mark foreign particles which are then taken up by the RES.The biological properties of PEG spacer elements used in accordance withthe invention may serve to increase the circulation time of the agent ina similar manner to that observed for PEGylated liposomes. Increasedcoupling efficiency to areas of interest may also be achieved usingantibodies bound to the terminii of PEG spacers.

Other representative spacer elements include structural-typepolysaccharides, storage-type polysaccharides, polyamino acids andmethyl and ethyl esters thereof, and polypeptides, oligosaccharides andoligonucleotides, which may or may not contain enzyme cleavage sites.

Preferred linking groups are derived from vector reactive groupsselected from but not limited to:—

a group that will react directly with carboxy, aldehyde, amine (NHR),alcohols, sulfhydryl groups, activated methylenes and the like, on thevector, for example, active halogen containing groups,

a group that can react readily with modified vector molecules containinga vector reactive group, i.e., vectors containing a reactive groupmodified to contain reactive groups, for example, by oxidation of thevector to an aldehyde or a carboxylic acid, and

a group that can be linked to the vector containing a reactive group, orto the modified vector as noted above by use of a crosslinking agent.

Preferred useful linking groups are derived from variousheterobifunctional cross-linking reagents such as those listed in thePierce Chemical Company Immunotechnology Catalog—Protein ModificationSection, (1995 and 1996).

In addition to the foregoing description, the linking groups, in wholeor in part, can also be comprised of and derived from complementarysequences of nucleotides and residues of nucleotides, both naturallyoccurring and modified, preferably non-self-associating oligonucleotidesequences.

Linking agents used in accordance with the invention will in generalbring about linking of vector to reporter or reporter to reporter withsome degree of specificity, and may also be used to attach one or moretherapeutically active agents.

Further examples of the linkers which may be used in the context of thecurrent application are given on pages 32-54 of WO98/47541 and thedisclosures made on these pages are incorporated herein by reference intheir entirety. It is hereby asserted that each and every linker or partthereof disclosed on the aforementioned pages is considered to be partof the description of the invention contained in this application.

Reporter

The reporter moieties in the contrast agents of the invention may be anymoiety capable of detection either directly or indirectly in an in vivodiagnostic imaging procedure, e.g. moieties which emit or may be causedto emit detectable radiation (e.g. by radioactive decay, fluorescenceexcitation, spin resonance excitation, etc.), moieties which affectlocal electromagnetic fields (e.g. paramagnetic, superparamagnetic,ferrimagnetic or ferromagnetic species), moieties which absorb orscatter radiation energy (e.g. chromophores and fluorophores), particles(including liquid containing vesicles), heavy elements and compoundsthereof, and moieties which generate a detectable substance, etc.

A very wide range of materials detectable by diagnostic imagingmodalities is known from the art and the reporter will be selectedaccording to the imaging modality to be used. Thus for example forultrasound imaging an echogenic material, or a material capable ofgenerating an echogenic material will normally be selected. The vectorsmay be coupled via a linker to a suitable lipid reporter/carrier forincorporation into a gas-filled microbubble. Such microbubbles may beused for targeting ultrasound imaging.

For X-ray imaging the reporter will generally be or contain a heavy atom(e.g. of atomic weight 38 or above). For MR imaging the reporter willeither be a non zero nuclear spin isotope (such as ¹⁹F) or a materialhaving unpaired electron spins and hence paramagnetic,superparamagnetic, ferrimagnetic or ferromagnetic properties. For lightimaging the reporter will be a light scatterer (e.g. a coloured oruncoloured particle), a light absorber or a light emitter. Formagnetometric imaging the reporter will have detectable magneticproperties; for electrical impedance imaging the reporter will affectelectrical impedance. For scintigraphy, SPECT, PET, etc., the reporterwill be a radionuclide.

Examples of suitable reporters are widely known from the diagnosticimaging literature, e.g. magnetic iron oxide particles, X-ray contrastagent containing vesicles, chelated paramagnetic metals (such as Gd, Dy,Mn, Fe etc.). See for example U.S. Pat. No. 4,647,447, PCT/GB97/00067,U.S. Pat. No. 4,863,715, U.S. Pat. No. 4,770,183, WO96/09840,WO85/02772, WO92/17212, PCT/GB97/00459, EP-A-554213, U.S. Pat. No.5,228,446, WO91/15243, WO93/05818, WO96/23524, WO96/17628, U.S. Pat. No.5,387,080, WO95/26205, GB9624918.0, etc. See also WO 98/47541 (pages63-66 and 70-86).

Particularly preferred as reporters are: chelated paramagnetic metalions such as Gd, Dy, Fe, and Mn, especially when chelated by macrocyclicchelant groups.

Stated generally, the reporter may be (1) a chelatable metal orpolyatomic metal-containing ion (i.e. TcO, etc), where the metal is ahigh atomic number metal (e.g. atomic number greater than 37), aparamagentic species (e.g. a transition metal or lanthanide), or aradioactive isotope, (2) a covalently bound non-metal species which isan unpaired electron site (e.g. an oxygen or carbon in a persistant freeradical), a high atomic number non-metal, or a radioisotope, (3) apolyatomic cluster or crystal containing high atomic number atoms,displaying cooperative magnetic behaviour (e.g. superparamagnetism,ferrimagnetism or ferromagnetism) or containing radionuclides, (4) achromophore (by which term species which are fluorescent orphosphorescent are included), e.g. an inorganic or organic structure,particularly a complexed metal ion or an organic group having anextensive delocalized electron system, or (5) a structure or grouphaving electrical impedance varying characteristics, e.g. by virtue ofan extensive delocalized electron system.

Examples of particular preferred reporter groups are described in moredetail below.

Chelated metal reporters: metal radionuclides, paramagnetic metal ions,fluorescent metal ions, heavy metal ions and cluster ions.

Preferred metal radionuclides include ⁹⁰Y, ^(99m)Tc, ¹¹¹In, ⁴⁷Sc, ⁶⁷Ga,⁵¹Cr, ^(177m)Sn, ⁶⁷Cu, ¹⁶⁷Tm, ⁹⁷Ru, ¹⁸⁸Re, ¹⁷⁷Lu, ²⁰³Pb and ¹⁴¹Ce.

Preferred paramagnetic metal ions include ions of transition andlanthanide metals (e.g. metals having atomic numbers of 6 to 9, 21-29,42, 43, 44, or 57-71), in particular ions of Cr, V, Mn, Fe, Co, Ni, Cu,La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu,especially of Mn, Cr, Fe, Gd and Dy, more especially Gd.

The metal ions are desirably chelated by chelant groups on the linkermoiety or in or on a particle, (e.g. a vesicle or a porous or non-porousinorganic or organic solid), in particular linear, macrocyclic,terpyridine and N₂S₂ chelants, such as for example DTPA, DTPA-BMA, EDTA,D03A and TMT. Further examples of suitable chelant groups are disclosedin U.S. Pat. No. 4,647,447, WO89/00557, U.S. Pat. No. 5,367,080, U.S.Pat. No. 5,364,613, etc.

The linker moiety or the particle may contain one or more such chelantgroups, if desired metallated by more than one metal species (e.g. so asto provide reporters detectable in different imaging modalities).

Other suitable residues of chelating agents comprise proteins modifiedfor the chelation of metals such as technetium and rhenium as describedin U.S. Pat. No. 5,078,985, the disclosure of which is herebyincorporated by reference.

Methods for metallating any chelating agents present are within thelevel of skill in the art. Metals can be incorporated into a chelantmoiety by any one of three general methods: direct incorporation,template synthesis and/or transmetallation. Direct incorporation ispreferred.

Thus it is desirable that the metal ion be easily complexed to thechelating agent, for example, by merely exposing or mixing an aqueoussolution of the chelating agent-containing moiety with a metal salt inan aqueous solution preferably having a pH in the range of about 4 toabout 11. The salt can be any salt, but preferably the salt is a watersoluble salt of the metal such as a halogen salt, and more preferablysuch salts are selected so as not to interfere with the binding of themetal ion with the chelating agent. The chelating agent-containingmoiety is preferrably in aqueous solution at a pH of between about 5 andabout 9, more preferably between pH about 6 to about 8. The chelatingagent-containing moiety can be mixed with buffer salts such as citrate,acetate, phosphate and borate to produce the optimum pH. Preferably, thebuffer salts are selected so as not to interfere with the subsequentbinding of the metal ion to the chelating agent.

In diagnostic imaging, the vector-linker-reporter (VLR) constructpreferably contains a ratio of metal radionuclide ion to chelating agentthat is effective in such diagnostic imaging applications. In preferredembodiments, the mole ratio of metal ion per chelating agent is fromabout 1:1,000 to about 1:1.

In radiotherapeutic applications, the VLR preferably contains a ratio ofmetal radionuclide ion to chelating agent that is effective in suchtherapeutic applications. In preferred embodiments, the mole ratio ofmetal ion per chelating agent is from about 1:100 to about 1:1. Theradionuclide can be selected, for example, from radioisotopes of Sc, Fe,Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Ru, Sn, Sr, Sm, Lu, Sb, W, Re,Po, Ta and Tl. Preferred radionuclides include ⁴⁴Sc, ⁶⁴Cu, ⁶⁷Cu, ²¹²Pb,⁶⁸Ga, ⁹⁰Y, ¹⁵³Sm, ²¹²Bi, ¹⁸⁶Re and ¹⁸⁸Re. Of these, especially preferredis ⁹⁰Y. These radioisotopes can be atomic or preferably ionic.

The following isotopes or isotope pairs can be used for both imaging andtherapy without having to change the radiolabeling methodology orchelator: ⁴⁷Sc₂₁; ¹⁴¹Ce₅₈; ¹⁸⁸Re₇₅; ¹⁷⁷Lu₇₁; ¹⁹⁹Au₇₉; ⁴⁷Sc₂₁; ¹³¹I₅₃;⁶⁷Cu₂₉; ¹³¹I₅₃ and ¹²³I₅₃; ¹⁸⁸Re₇₅ and ^(99m)Tc₄₃; ⁹⁰ Y₃₉ and ⁸⁷Y₃₉;⁴⁷Sc₂₁ and ⁴⁴Sc₂₁; ⁹⁰Y₃₉ and ¹²³I₅₃; ¹⁴⁶Sm₆₂ and ¹⁵³Sm₆₂; and ⁹⁰Y₃₉ and¹¹¹In₄₉.

The linker moiety may also allow for coupling to a plurality of chelantgroups. The chelant moieties within such a polychelant linker may beattached via backbone functionalization of the chelant or by utilizationof one or more of the metal co-ordinating groups of the chelant or byamide or ether bond formation between acid chelant and an amine orhydroxyl carrying linker backbone, e.g. as in polylysine-polyDTPA,polylysine-polyDOTA and in the so-called magnifier polychelants, ofPCT/EP96/00565. Such polychelant linkers may be conjugated to one ormore vector groups either directly (e.g. utilizing amine, acid orhydroxyl groups in the polychelant linker) or via a bifunctional linkercompound as discussed above for monochelant linkers.

Where the chelated species is carried by a particulate (or molecularaggregate, e.g. vesicular) linker, the chelate may for example be anunattached mono or polychelate (such as Gd DTPA-BMA or Gd HP-DO3A)enclosed within the particle or it may be a mono or polychelateconjugated to the particle either by covalent bonding or by interactionof an anchor group (e.g. a lipophilic group) on the mono/polychelatewith the membrane of a vesicle (see for example PCT/GB95/02378).

Preferred non-metal atomic reporters include radioisotopes such as ¹²³Iand ¹³¹I as well as non zero nuclear spin atoms such as ¹⁹F, and heavyatoms such as I.

Such reporters, preferably a plurality thereof, e.g. 2 to 200, may becovalently bonded to a linker backbone, either directly usingconventional chemical synthesis techniques or via a supporting group,e.g. a triiodophenyl group.

In an embodiment of this invention, the use of radioisotopes of iodineis specifically contemplated. For example, if the vector or linker iscomprised of substituents that can be chemically substituted by iodinein a covalent bond forming reaction, such as, for example, substituentscontaining hydroxyphenyl functionality, such substituents can be labeledby methods well known in the art with a radioisotope of iodine. Theiodine species can be used in therapeutic and diagnostic imagingapplications. While, at the same time, a metal in a chelating agent onthe same vector-linker can also be used in either therapeutic ordiagnostic imaging applications. As with the metal chelants discussedabove, such metal atomic reporters may be linked to the linker orcarried in or on a particulate linker, e.g. in a vesicle (see WO95/26205and GB 9624918.0).

Linkers of the type described above in connection with the metalreporters may be used for non-metal atomic reporters with the non-metalatomic reporter or groups carrying such reporters taking the place ofsome or all of the chelant groups.

Preferably the V-L-R agents of the invention will have the receptortargetting vectors coupled directly or indirectly to a reporter, e.g.with covalently bound iodine radioisotopes, with metal chelates attacheddirectly or via an organic linker group or coupled to a particulatereporter or linker-reporter, e.g. a superparamagnetic crystals(optionally coated, e.g. as in PCT/GB97/00067), or a vesicle, e.g. aniodinated contrast agent containing micelle or liposome.

Put briefly, for the imaging modalities of MRI, X-ray, light imaging,nuclear imaging, magnetotomography and electrical impedance tomography,the favoured reporters may be as follows:

MRI Superparamgnetic iron oxide particles, in general having a particlesize smaller than about 80 nm and especially those with a size less than20 nm. In particular iron oxides coated with various coating materialssuch as polyelectrolytes, PEG, starch and hydrolyzed starch arepreferred. Paramagnetic metal substances including both chelates andparticulate materials are also useful. Light imaging Any light imagingreporter group. The focus should be on substances absorbing in the nearinfrared range. Nuclear medicine Radioactive chelates comprising “Tc or¹¹¹In as well as direct radiolabelled vectors having radiolabelledhalogens substituents such as ¹²³I, ¹²⁵I, ¹³¹I, ⁷⁵Br or ⁷⁷Br.Magnetotomography Superparamagnetic iron oxide particles as describedabove. Electrical Polyionic species, e.g. polymers impedance with ionicgroups in the repeat tomography units.

A preferred embodiment of the invention relates to a radiolabelled agentof general formula (I), particularly for use in tumour imaging.

The diagnostic agents of the invention may be administered to patientsfor imaging in amounts sufficient to yield the desired contrast with theparticular imaging technique. Where the reporter is a metal, generallydosages of from 0.001 to 5.0 mmoles of chelated imaging metal ion perkilogram of patient bodyweight are effective to achieve adequatecontrast enhancements. For most MRI applications preferred dosages ofimaging metal ion will be in the range of from 0.02 to 1.2 mmoles/kgbodyweight while for X-ray applications dosages of from 0.05 to 2.0mmoles/kg are generally effective to achieve X-ray attenuation.Preferred dosages for most X-ray applications are from 0.1 to 1.2 mmolesof the lanthanide or heavy metal compound/kg bodyweight. Where thereporter is a radionuclide, dosages of 0.01 to 100 mCi, preferably 0.1to 50 mCi will normally be sufficient per 70 kg bodyweight. Where thereporter is a superparamagnetic particle, the dosage will normally be0.5 to 30 mg Fe/kg bodyweight.

The dosage of the compounds of the invention for therapeutic use willdepend upon the condition being treated, but in general will be of theorder of from 1 pmol/kg to 1 mmol/kg bodyweight.

The compounds according to the invention may be formulated foradministration using physiologically acceptable carriers or excipientsin a manner fully within the skill of the art. For example, thecompounds, optionally with the addition of pharmaceutically acceptableexcipients, may be suspended or dissolved in an aqueous medium, with theresulting solution or suspension then being sterilized.

The agents of formula I may be therapeutically effective in thetreatment of disease states as well as detectable in in vivo imaging.Thus for example the vector of the VLR compound may have therapeuticefficacy, e.g. by virtue of the radiotherapeutic effect of aradionuclide reporter, the efficacy in photodynamic therapy of achromophore (or fluorophore) reporter or the chemotherapeutic effect ofthe vector moiety.

Use of the agents of formula I in the manufacture of therapeuticcompositions and in methods of therapeutic or prophylactic treatment ofthe human or non-human animal body are thus considered to representfurther aspects of the invention.

Viewed from a further aspect the invention provides the use of an agentof formula I for the manufacture of a contrast medium for use in amethod of diagnosis involving administration of said contrast medium toan animate subject and generation of an image of at least part of saidsubject.

Viewed from a still further aspect the invention provides a method ofgenerating an image of an animate human or non-human (preferablymammalian or avian) animal subject involving administering a contrastagent to said subject, e.g. into the vascular system and generating animage of at least a part of said subject to which said contrast agenthas distributed, e.g. by X-ray, MR, ultrasound, scintigraphy, PET,SPECT, electrical impedance, light or magnetometric imaging modalities,characterised in that as said contrast agent is used an agent of formulaI.

Viewed from a further aspect the invention provides a method ofmonitoring the effect of treatment of a human or non-human animalsubject with a drug to combat a condition associated with angiogenesis,e.g. a cytotoxic agent, said method involving administering to saidsubject an agent of formula I and detecting the uptake of said agent byendothelial cell receptors, in particular αvβ3 receptors, saidadministration and detection optionally but preferably being effectedrepeatedly, e.g. before, during and after treatment with said drug.

Viewed from a yet further aspect the invention provides a process forthe preparation of an agent of formula I, said process comprising theconjugation of a vector V to a compound detectable in a diagnosticimaging procedure or a chelant compound and if necessary metallatingchelant groups in the resultant conjugate with a metal ion detectable ina diagnostic imaging procedure.

Viewed from yet another aspect the invention provides a process for thepreparation of an agent of formula I, for therapeutic treatment, saidprocess comprising the conjugation of a vector V to a compound to betherapeutically effective in the treatment of disease states.

The vectors of the present invention can be synthesised using all theknown methods of chemical synthesis but particularly useful is thesolid-phase methodology of Merrifield employing an automated peptidesynthesiser (J. Am. Chem. Soc., 85: 2149 (1964)). Vectors containingmultiple disulphide bridges are synthesised using differential cysteineprotecting groups so that no ambiguity exists as to the final foldedform of the vector. The peptides and peptide chelates may be purifiedusing high performance liquid chromatography (HPLC) and characterised bymass spectrometry and analytical HPLC before testing in the in vitroscreen.

There follows a series of non-limitive examples:

Example 1 Synthesis of compound 1

Synthesis of technetium chelate—Pn216a) Chloro-nitroso intermediate (3-chloro-3-methyl-2-nitrosobutane)

A mixture of 2-methylbut-2-ene (18.5 mL) and iso-amyl nitrate (19.5 mL)was stirred, cooled to −10° C. and concentrated hydrochloric acid (17.5mL) added carefully to maintain the temperature below 0° C. The reactionwas stirred at this temperature for 30 minutes. The precipitate formedwas collected by filtration, washed 4×5 mL of ethanol (−20° C.) anddried in vacuo to give 3-chloro-3-methyl-2-nitrosobutane as a whitesolid.

b)Pn216—(3,3,11,11-tetramethyl-7-aminoethyl-4,7,10,triazamidecane-2,12-dionedioxime)

To a solution of tris-(2-aminoethyl) amine in acetonitrile (20 mL) wasadded sodium bicarbonate (2.2 g, 26 mmol). A solution of3-chloro-3-methyl-2-nitrosobutane (1.8 g, 13 mmol) in dry acetonitrilewas added slowly at 0° C. The reaction mixture was left to stir at roomtemperature for 4 hours and then filtered. The filtrant was washed withacetonitrile and the filtrate evaporated. The crude product wasdissolved in acetonitrile and purified by HPLC to afford Pn216. Yield0.88 g, 19%

c) Synthesis of Pn216-succinic acid intermediate:

Succinic anhydride (100)Pn216 (358)Tetrafluorothiophenol (182)DCCI (206)

Pn216 (0.5 g, 1.4 mmol) was dissolved in DMF (5 mL) and succinicanhydride (0.015 g, 1.5 mmol) in DMF (10 mL) added portionwise withstirring. The reaction was left stirring for 16 hours to afford completeconversion to the desired product. The pure acid was obtained followingHPLC chromatography in good yield.

d) Synthesis of the tetrafluorothiophenol ester derivative ofPn216-succinic acid

HATU [0-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate]—Mwt=380

Pn216—NH—CO—(CH2)₂-COOH—Mwt=458

NMM—N-methylmorpholine—Mwt=101

TFTP—tetrafluorothiophenol—Mwt=182

To Pn216 acid (10 mg, 0.022 mmol) in DMF (1.0 mL) was added HATU (8.3mg, 0.022 mmol) and NMM (0.007 mL, 0.066 mmol). The mixture was stirredfor 5 minutes then TFTP (0.022 mmol, 4 mg) added. The solution wasstirred for 30 minutes then the reaction mixture was diluted with 20%acetonitrile/H₂O (3 mL) and the product purified by reverse-phasechromatography yielding 6 mg of the desired product followingfreeze-drying.

e) Synthesis of peptide vectorNH₂-Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly-OH (Seg. ID 1) withdisulphide bonds connecting Cys 2 and 4; Cys 8 and 10

The peptide was synthesised on a ABI 433A automatic peptide synthesiserstarting with Fmoc-Gly Wang resin (Novabiochem) on a 0.1 mmol scaleusing 1 mmol amino acid cartridges. Cysteine residues 2 and 4 wereS-protected using trityl protection while 8 and 10 were protected withacetamidomethyl (Acm) protection. The amino acids were pre-activatedusing HBTU before coupling. The simultaneous removal of peptide andside-chain protecting groups (except Acm) from the resin was carried outin TFA containing TIS (5%), H₂0 (5%) and phenol (2.5%) for two hours.

After work-up 100 mg of partially protected crude peptide was obtained(Analytical HPLC: Gradient, 0-30% B over 20 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, VYDAC C18 218TP54; detection, UV 214 nm;product retention time, 16.7 min). An aliquot of crude product (25 mg)was then purified by Prep. HPLC Vydac column yielding 12.5 mg of purepartially protected peptide.

The first disulphide bond between Cys2 and Cys4 was formed by dissolvingthe pure intermediate in 20 mL of 2.5% DMSO/TFA solution. After 40minutes a new peak had appeared corresponding to the oxidised product.To the peptide solution was then added anisole (0.02 mL) and thesolution warmed to 60° C. for 50 minutes. Excess TFA was then removed invacuo and the product precipitated following addition of diethyl ether.A further Prep. HPLC step was carried out and pure product collected andfreeze-dried. MALDI-TOF analysis was used to confirm molecular weightand a co-injection with the two other possible disulphide isomersperformed to confirm identity.

f) Synthesis of compound 1:

Peptide from section e) above is dissolved together with Pn216 activeester from section d) above in DMF in a 1:2 ratio (w:w). The reaction isleft stirring for 2 days then the mixture is diluted with water and thedesired product purified out by reverse-phase HPLC.

Example 2 [Cys^(2-4, 8-10)] analogues

a) Synthesis of C1CH₂CO-Lys-Asp-Cys-Arg-Gly-Asp-Cys(tBu)-Phe-Cys(tBu)-Gly-Gly-OH (Seg. ID 2)

The peptide was synthesised on an ABI 433A automatic peptide synthesiserstarting with Fmoc-Gly-Wang resin on a 0.25 mmol scale using 1 mmolamino acid cartridges. The amino acids were pre-activated using HBTUbefore coupling. Final N-terminal chloroacetylation was accomplishedusing a solution of chloroacetic anhydride in DMF for 30 min.

The simultaneous removal of peptide and side-chain protecting groups(except tBu) from the resin was carried out in TFA containing TIS (5%),H₂0 (5%) and phenol (2.5%) for two hours.

After work-up 260 mg of crude peptide was obtained (Analytical HPLC:Gradient, 5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA;column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection,UV 214 nm; product retention time, 6.5 min. Further productcharacterisation was carried out using mass spectrometry: Expected, M+Hat 1348.5, found, at 1348.5).

b) Synthesis of cyclo[CH₂CO-Lys-Asp-Cys]-Arg-Gly-Asp-Cys (tBu)-Phe-Cys(tBu)-Gly-Gly-OH

100 mg ClCH₂CO-Lys-Asp-Cys-Arg-Gly-Asp-Cys(tBu)-Phe-Cys(tBu)-Gly-Gly-OH(Seq. ID 2) was dissolved in water/acetonitrile. The mixture wasadjusted to pH 8 with ammonia solution and stirred for 24 hours.

After work-up crude peptide was obtained (Analytical HPLC: Gradient,5-50% B over 10 min where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA; column,Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2 mL/min; detection, UV 214nm; product retention time, 6.32 min. Further product characterisationwas carried out using mass spectrometry: Expected, M+H at 1312.5, found,at 1312.6).

c) Synthesis of [Cys⁷⁻⁹]cyclo[CH₂CO-Lys-Asp-Cys]-Arg-Gly-Asp-Cys-Phe-Cys-Gly-Gly-OH

40 mg ofcyclo[CH₂CO-Lys-Asp-Cys]-Arg-Gly-Asp-Cys(tBu)-Phe-Cys(tBu)-Gly-Gly-OHwas treated with a solution of anisole (200 μL), DMSO (1 mL) and TFA (50mL) for 30 min following which the TFA was removed in vacuo and thepeptide precipitated by the addition of diethyl ether.

Purification by preparative HPLC (Phenomenex Luna 5μ C18 (2) 250×21.20mm column) of the crude material (40 mg) was carried out using 0-30% B,where A=H₂O/0.1% TFA and B=CH₃CN/0.1% TFA, over 40 min at a flow rate of10 mL/min. After lyophilisation 14.3 mg of pure material was obtained(Analytical HPLC: Gradient, 0-30% B over 10 min where A=H₂O/0.1% TFA andB=CH₃CN/0.1% TFA; column, Phenomenex Luna 3μ C18 (2) 50×4.6 mm; flow, 2mL/min; detection, UV 214 nm; product retention time, 6.10 min. Furtherproduct characterisation was carried out using mass spectrometry:Expected, M+H at 1198.4, found, at 1198.5).

d) Conjugation of [Cys⁷⁻⁹]cyclo[CH₂CO-Lys-Asp-Cys]-Arg-Gly-Asp-Cys-Phe-Cys-Gly-Gly-OH andPn216-succinic acid

[Cys⁷⁻⁹] cyclo[CH₂CO-Lys-Asp-Cys]-Arg-Gly-Asp-Cys-Phe-Cys-Gly-Gly-OH,Pn216 chelate active ester and N-methylmorpholine is dissolved in DMF.The mixture is stirred until complete conjugation is observed by RP-HPLCmonitoring.

Purification by preparative RP-HPLC of the reaction mixture is carriedout to afford pure material following lyophilisation. Productcharacterisation is carried out using RP-HPLC and mass spectrometry.

1. A compound of general formula (I)

wherein r=0 or 1 p=0 or 1 and r+p=1 when r=0 the R₁ is —(CH₂)_(n)—CO— or—(CH₂)_(n)—C₆H₄—CO— where n=1, 2, 3, 4 or 5, when r=1 then R₁ is one ormore bridge-forming amino acids, X_(i)=an amino acid functionalized witha chelate capable of binding a reporter suitable for in vivo imaging, X₂and X₄ are independently cysteine, X₃ is N-methylarginine, X₅ is ahydrophobic amino acid, X₆ is cysteine, X₇ is a bond or linkeroptionally allowing for labeling with chelates as defined by X₈, andoptionally comprising one or more ethylene glycol units or any otherspacer component, and X₈ is a chelate capable of binding a metalradionuclide or any other reporter suitable for in vivo imaging, or isNH₂ or is absent, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, and one of thebridges, between R₁ and X₂ or between X₄ and X₆, comprises a disulphidebond.
 2. A compound as claimed in claim 1 wherein R₁ is cysteine.
 3. Acompound as claimed in claim 1 wherein X₆ is cysteine.
 4. A compound asclaimed in claim 1 wherein X₅ is tyrosine, iodotyrosine, diiodotyrosineor naphthylalanine.
 5. A pharmaceutical composition comprising aneffective amount of a compound of general formula (I) of claim 1 or anacid addition salt thereof to enhance image contrast for in vivo imagingor for therapeutic treatment, together with one or more pharmaceuticallyacceptable adjuvants, excipients or diluents.
 6. A method of generatingenhanced images of a human or non-human animal body which comprisesadministering to said body a pharmaceutical composition as claimed inclaim 5 and generating an image of at least a part of said body.
 7. Amethod of monitoring the effect of treatment of a human or non-humananimal subject with a drug to combat a condition associated withangiogenesis which comprises administering to said subject a compound offormula I, of claim 1, and detecting the uptake of said compound, saidadministration and detection optionally but preferably being effectedrepeatedly, e.g. before, during and after treatment with said drug. 8.The method of claim 7, wherein said administration and detection areeffected repeatedly.
 9. The method of claim 8, wherein saidadministration and detection are effected before, during and aftertreatment with said drug.
 10. The method of claim 7, wherein thecompound of formula I is prepared by the step comprising metallatingchelant groups to the compound of formula I with a metal ion detectablein a diagnostic imaging procedure.